458 Original article
Tumor necrosis factor-a –308G/A gene polymorphism in Egyptian children with immune thrombocytopenic purpura Maha H. El Sissya, A.H. El Sissyb and Sherif Elanwaryc Immune thrombocytopenic purpura (ITP) is an autoimmune disease characterized by increased platelet destruction. Although the cause of ITP remains unclear, it is accepted that both environmental and genetic factors play an important role in the development of the disease. Children with ITP have a T-helper 1-type cytokine pattern with elevated levels of tumor necrosis factor-alpha (TNF-a) as in most autoimmune diseases. Researchers have shown that polymorphism in the TNF-a gene at position S308 affects gene transcriptions with increased TNF-a production. The current case–control study aimed at detecting the frequency of TNF-a S308G/A gene polymorphism as genetic markers in Egyptian children with ITP, and to clear out their possible role in choosing the treatment protocols of therapy, using PCR restriction fragment length polymorphism assay. Ninety-two ITP patients and 100 age and sex-matched healthy controls were recruited in the study. The results obtained revealed that the frequency of TNF-a S308A/A homotype in ITP patients was significantly higher than that of the controls, and conferred almost six-fold increased risk of ITP acquisition.
The polymorphic A allele frequency was significantly higher in ITP patients than in the controls, conferring almost two-fold increased ITP risk. In conclusion, our study suggests the possibility that TNF-a S308 gene polymorphism may contribute to the susceptibility of childhood ITP in Egyptian children. Blood Coagul Fibrinolysis 25:458–463 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins.
Introduction
(1) Newly diagnosed ITP: cases within 3 months of diagnosis. (2) Persistent ITP: they are the cases between 3 and 12 months of diagnosis and they are the patients who fail to maintain a complete response with treatment or who fail to reach spontaneous remission. (3) Chronic ITP: it continues for more than 12 months. (4) Refractory ITP: the following three criteria are required: (a) loss of remission or failure of remission after splenectomy (b) requirement of treatment to minimize the risk of clinically significant hemorrhage (c) all the other causes of primary ITP and thrombocytopenia have to be eliminated.
Immune thrombocytopenic purpura (ITP) is one of the most frequent causes of thrombocytopenia and is characterized by destruction of autoantibody-mediated platelets. The pathophysiology of ITP is complex and includes antibodies, cytokines, antigen-presenting cells, co-stimulatory molecules, T and B lymphocytes. When autoreactive B lymphocytes produce antiplatelet antibodies, T lymphocytes play an important role in this autoimmune process [1,2]. ITP is an acquired autoimmune disorder and the most common cause of isolated thrombocytopenia in children [3]. Immune thrombocytopenic purpura is caused by the production of antiplatelet antibodies. These autoantibodies opsonize platelets for splenic clearance; resulting in low levels of circulating platelets [4]. These antibodies may also impair platelet production, creating a dual cause of thrombocytopenia [5]. Complete recovery is usually expected within a few days or weeks, regardless of treatment. Nevertheless, therapy is given to patients with ITP because of occasional major bleeding episodes. In approximately 30% of cases, the disease will follow a chronic course with prolonged and recurrent thrombocytopenia [6]. Akalin and Murphy [7] in 2001 classified ITP into the following phases: 0957-5235 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
Blood Coagulation and Fibrinolysis 2014, 25:458–463 Keywords: tumor necrosis factor-alpha, immune thrombocytopenic purpura, genetic polymorphism a
Clinical Pathology Department, bClinical Pathology Department, National Cancer Institute and cPediatric Department, Cairo University, Cairo, Egypt Correspondence to Maha H. El Sissy, MD, Kasr Al aini Hospitals, Cairo University, Cairo, Egypt Tel: +002 01229193373; e-mail: [email protected] Received 9 October 2013 Revised 25 December 2013 Accepted 26 December 2013
It was previously reported that there was an association between the cytokine gene polymorphisms affecting the cytokine production and secretion and infectious diseases, allergic diseases, autoimmune diseases, and malignant diseases, both at the stage of formation of disease and in the course of disease and their responses to treatment [7]. Population-based studies have shown that ITP has an incidence of up to 6.4 per 100 000 children and 3.3 per 100 000 adults per year [8]. DOI:10.1097/MBC.0000000000000089
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Tumor necrosis factor-alpha 308G/A in Egyptian children with ITP El Sissy et al. 459
Children with ITP have a T-helper 1 (Th1)-type cytokine pattern with elevated levels of tumor necrosis factoralpha (TNF-a), as in most autoimmune diseases [9]. Researchers have shown that polymorphism in the TNF-a gene at position 308 affect gene transcription with increased TNF-a production [10]. The transcriptionally more active allele of TNF-a (allele 2 of 308) was less frequently observed in pediatric chronic ITP patients than in healthy controls [11]. Tumor necrosis factor-alpha is a pleiotropic cytokine produced primarily by macrophages and T cells, and has a range of inflammatory and immunomodulatory activity [12]. Polymorphisms of TNF-a promoter are associated with high levels of TNF-a and have been studied as a determinant of susceptibility to numerous diseases [13]. A study that examined inflammatory cytokines and Fcg receptor (FcgR) polymorphisms in chronic childhood ITP reported that polymorphisms in two low-affinity FcgR genes (IIIA and IIIB) and two pro-inflammatory cytokine genes [TNF-a and lymphotoxin-alpha (LTA)] were associated with chronic childhood ITP [11]. A single-base polymorphism within the promoter region of TNF gene at nucleotide position 308 results in two allelic forms: one with guanine, TNF-G allele (TNF1 allele), and the other, in which guanine is substituted by adenosine, TNF-A allele (TNF2 allele). TNF-A allele has been found to correlate with enhanced spontaneous or stimulated TNF production in vivo and in vitro [14]. The current case–control study aimed at detecting the frequency of TNF-a 308G/A gene polymorphism in Egyptian children with ITP as genetic markers for ITP risk, and to clear out their possible role in choosing the treatment protocols of ITP patients.
Patients and methods The present study was conducted on 92 ITP patients (12 acute and 80 chronic) attending the Haematology Clinic of the Children Hospital, Faculty of Medicine, Cairo University, as well as 100 age and sex-matched healthy children as a control group; all patients and control groups gave a written consent accepting research aid and our case–control study was approved by the ethical committee of the Clinical Pathology Department, Cairo University. Diagnosis of ITP was based on proper history taking with special attention to history of intake of medications known to induce signs of ITP as petechiae, ecchymosis or mucous membrane bleeding, and to exclude signs of concomitant autoimmune disorders such as juvenile rheumatoid arthritis, systemic lupus erythematosus (SLE), and vitiligo. Laboratory assessment included complete blood count revealing isolated thrombocytopenia) platelet count 12 months’ duration) [17]. The study design was approved by the Scientific Research Committee of Clinical Pathology and Pediatrics Departments, Faculty of Medicine, Cairo University. All parents and/or surrogates were informed about the aims of the study. Formal written consents were obtained from the caregivers of the participants who agreed to their child’s participation in the study.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
460 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 5
Detection of tumor necrosis factor-alpha S308 genetic polymorphism by PCR restriction fragment length polymorphism assay
Five milliliters venous blood was withdrawn under complete aseptic conditions from all participants on EDTA. Samples were either stored in the same vacutainer at 208C or used directly within 24 h for DNA extraction. DNA was extracted from whole blood using AxyPrep Blood Genomic DNA Miniprep Kit (Axygen Biosciences, Union City California, USA). Genotyping was based upon the methods described by Jrad et al. [18] as the target genes were amplified by PCR then restriction with the endonuclease NcoI. All PCR reactions were performed in a total volume of 25 ml containing 12.5 ml Master mix (Bioron GmbH, Ludwigshafen Germany), 1 ml forward primer (25 pmol), 1 ml reverse primer (25 pmol), 5.5 ml nuclease-free water and 5 ml genomic extracted DNA. For TNF-a 308 genotyping, the following primers were used: forward primer 50 -AGGCAATAGGTTTTGAGG GCCAT-30 , and reverse primer 50 -TCCTCCCTGCT CCGATTCCG-30 (Fermentas, Lithuania). The thermocycler program applied was heating at 958C for 5 min, followed by 29 cycles of denaturation at 958C for 30 s, annealing at 608C for 30 s, and extension at 728C for 45 s. A final extension step was carried out at 728C for 10 min. The PCR product (107 bp) was digested with NcoI (Fermentas, Cat. No. #ER0571). The product was visualized by 3% agarose gel electrophoresis stained with ethidium bromide under ultraviolet (UV) light. A DNA molecular weight marker was also run to identify the site of bands. The presence of NcoI restriction site in the mutant allele of TNF-a (A allele) was indicated by the cleavage of the 107 bp amplicon to yield two fragments of 87 and 20 bp (Fig. 1).
data were expressed as mean and SD or median and range as appropriate. Qualitative data were expressed as frequency and percentage. Chi-squared test (Fisher’s exact test) was used to examine the relation between qualitative variables. For quantitative data, comparison between two groups was done using either Student’s t-test or Mann–Whitney test (nonparametric t-test). Comparison between three groups was done using Kruskal–Wallis test [nonparametric analysis of variance (ANOVA)]. Odds ratio (OR) and 95% confidence intervals (CIs) were used for risk estimation. Kappa test was used to evaluate agreement between two genotypes. A P-value less than 0.05 was considered significant.
Results The study included 92 ITP cases. They were 48 females (52.2%) and 44 males (47.8%). Their ages ranged between 1 and 14 years with a mean value of age 8.26 4.49 years. Twelve patients (12/92 13.1%) were acute and 80 of 92 (86.9%) were chronic cases; clinical and laboratory data of ITP patients are summarized in Table 1. One hundred age and sex-matched unrelated healthy individuals were included in the current study as a control group. They were 45 females (45%) and 55 males (55%). Their ages ranged between 1 and 13 years with mean value of 6.18 3.494 years. Indirect antiplatelet antibodies were detected in three patients with acute ITP (3/12–25%) and 20 patients (20/80–25%) with chronic ITP, revealing no statistically significant difference between acute and chronic cases.
Data were analyzed using IBM SPSS advanced statistical version 20 (SPSS Inc., Chicago, Illinois, USA). Numerical
The frequency of TNF-a 308A/A homotype in ITP patients was significantly higher than that of the controls, and conferred almost six-fold increased risk of ITP acquisition (OR 6.5, 95% CI 2.54–16.65). The frequency of the combined polymorphic genotypes – TNF-a 308G/A heterotype and A/A homotype – was
Fig. 1
Table 1
Statistical analysis
Descriptive data of ITP patients
Item Sex Bleeding Spleen
Degree of anemia
Degree of thrombocytopenia
Identification of the TNF-308 genotypes by agarose gel electrophoresis. M: 50–1000 Ladder size marker. The 20-bp band is not visible in this photograph. Lanes 3 and 5: TNF-308 GG genotype (wild type) showing a 107-bp band. Lane 1: TNF-308 AA homotype showing 87-bp band. Lanes 2 and 4: TNF-308 GA heterotype showing 87 and 107-bp bands. TNF, tumor necrosis factor.
History of viral infections or immunization Family history Type of ITP Therapy
Male Female Positive Enlarged Normal Splenectomy Mild Moderate Marked Hb: 11gm% Moderate Marked Positive Negative Negative Acute Chronic Steroids
Frequency
Percentage
44/92 48/92 92/92 4/92 79/92 9/92 80/92 8/92 3/92 1/92 6/92 86/92 31/92 61/92 92/92 12/92 80/92 92/92
47.8 52.2 100 4.3 85.9 9.8 87 8.7 3.3 1.1 6.5 93.5 33.7 66.3 100 13 87 100
Hb, haemoglobin; ITP, immune thrombocytopenic purpura.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Tumor necrosis factor-alpha 308G/A in Egyptian children with ITP El Sissy et al. 461
Table 2
Genotypes and alleles frequency of TNF-a S308 polymorphism in ITP cases and controls
TNF-a 308 Wild genotype (G/G) Heterotype (G/A) Homotype (A/A) Combined polymprphic genotypes (G/A–A/A) G wild-type allele A polymorphic allele
Cases (n ¼ 92)
Control (n ¼ 100)
43/92 22/92 27/92 49/92 86 (0.54) 98 (0.38)
63/100 31/100 6/100 37/100 128 (0.80) 72 (0.195)
OR (95% CI) 1.439 0.849 6.5 1.94 1.94
(1.046–1.981) (0.640–1.125) (2.54–16.65) (1.09–3.45) (1.09–3.45)
P-value 0.017 0.175 0.001 0.024 0.001
CI, confidence interval; ITP, immune thrombocytopenic purpura; OR, odds ratio; TNF-a, tumor necrosis factor-alpha.
significantly higher in ITP cases than in controls (OR 1.94, 95% CI 1.09–3.45, P-value 0.024).
platelet antigen (HPA) systems, were associated with the development of ITP and response to treatment [20].
Also the polymorphic A allele frequency was significantly higher in ITP patients than the controls conferring almost two-fold increased ITP risk (OR 1.94, 95% CI 1.09–3.45) (Table 2), suggesting its possible role as a genetic risk factor for tendency of childhood ITP.
Tumor necrosis factor-alpha is a critical cytokine in the inflammatory response to infection. Accordingly, any genetic variability in the production of TNF-a after an infectious stimulus could have a significant impact on the degree of inflammatory response and therefore potentially influence the clinical outcome [21].
Statistical comparison between ITP patients with the wild genotype and those with polymorphic genotypes of TNFa 308 gene revealed that the frequency of the polymorphic genotype was significantly lower in males and lactate dehydrogenase level was significantly lower among ITP patients harboring the polymorphic A allele. Otherwise, there was no statistically significant difference noticed between the two patient groups, as regards the degree of thrombocytopenia, bleeding, splenomegaly, or their response to treatment (data not shown). The frequency of TNF-a 308G/A heterotype and A/A homotype in acute ITP patients was not significantly different from chronic ITP (OR 1.481, 95% CI 0.604– 3.635 and OR 0.833, 95% CI 0.296–2.347, respectively); also, the polymorphic A allele frequency was not significantly different in acute than chronic ITP patients (Table 3).
Discussion Although the cause of ITP remains unclear, it is generally accepted that both environmental and genetic factors play an important role in the development of the disease [19]. Many studies have focused on the association between some cytokine gene polymorphisms and susceptibility to ITP. Studies that investigated genetic risk factors in the pathogenesis of ITP reported that polymorphisms of human leucocyte antigen class II antigens, TNF-a, transforming growth factor-b, and human Table 3
Significant evidence supports the biologic importance of polymorphisms within the TNF-a promoter region. A guanine (G) to adenine (A) transition at TNF-a 308 is perhaps the best studied cytokine polymorphism and the one for which the best evidence of functional significance exists [22]. Stimulation studies in healthy volunteers suggested that carriage of the TNF-a 308 A allele is associated with significantly greater TNF-a production [23] and TNF-a mRNA transcription [24]. Tumor necrosis factor-alpha is a potent immunomediator and pro-inflammatory cytokine shown in the pathogenesis of many human diseases. In their meta-analysis, Teuffel et al. [25] indicated that TNF-a 308A/G polymorphism was associated with sepsis. Settin et al. [26] found that the frequency of TNF-a 308G/A genotype increased in patients with psoriasis and they determined the G/G frequency to be low. Gambhir et al. [27] reported that the frequency of TNF-a 308 A genotype was lower in 222 cases with rheumatoid arthritis in the northern section of India than in 208 healthy control cases, and that the presence of this genotype was protective against the disease. In their study in Taiwan, Lin et al. [28] detected the frequency of allele A in TNF-a 308 polymorphisms as significantly high in cases with malar rash, discoid rash, photosensitivity, oral ulcer, and serositis compared to the control cases in SLE patients, and they demonstrated that in the Taiwanese patient population,
Risk genotypes and alleles frequency of TNF-a S308 polymorphism in acute and chronic ITP patients
TNF-a 308 Wild genotype (G/G) Heterotype (G/A) Homotype (A/A) G wild-type allele A polymorphic allele
Acute ITP (n ¼ 12)
Chronic ITP (n ¼ 80)
5/12 4/12 3/12 10 (0.58) 14 (0.41)
38/80 18/80 24/80 76 (0.58) 84 (0.41)
OR (95% CI) 1.111 1.481 0.833 1.010
(0.659–1.872) (0.604–3.635) (0.296–2.347) (0.608–1.689)
P-value 0.475 0.311 0.509 0.569
CI, confidence interval; ITP, immune thrombocytopenic purpura; OR, odds ratio; TNF-a, tumor necrosis factor-alpha.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
462 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 5
TNF-a 308 polymorphism played a role in susceptibility to the formation of SLE. Vural et al. [29] showed that the frequency of TNF-a 308 gene polymorphisms increased 6.5-fold in sporadic late-onset Alzheimer’s disease compared to the controls. Cerri et al. [30] demonstrated that in elderly patients with major depression, TNF-a 308G/A genotype contributed to the development of major depression. To the best of our knowledge, the role of TNF-a 308A/ A homotype polymorphism in the development of ITP has not been previously studied in Africans. In the present study, the genotype frequency of TNF-a 308 mutant genotypes G/A heterotype and A/A homotype polymorphism was evaluated in terms of their correlation with susceptibility to the development of ITP. In the current study, genotyping of TNF-a 308 gene by PCR restriction fragment length polymorphism (PCR-RFLP) assay revealed that the wild-type alleles were detected in 46.8%, whereas the mutant genotypes of TNF-a 308G/A heterotype and A/A homotype were detected in 23.9 and 29.3% of the patients, respectively. The frequency of the mutant genotypes in Egyptian ITP patients were different to that reported in the study by Okulu et al. [31] (36%) in Turkish populations. The frequency of TNF-a 308G/A heterotype in ITP patients was near to that of the control individuals (22 versus 31%). This was not in accordance with the study by Okulu et al. who reported (36 versus 22.9%) ITP patients versus control individuals, respectively. The frequency of TNF-a 308A/A homotype in ITP patients was not close to that of the control individuals (27 versus 6%). This was not in accordance with the study by Okulu et al. who reported (18 versus 22.9%) ITP patients versus control individuals, respectively. No statistically significant difference was noticed between ITP patients harboring the normal or polymorphic TNF-a 308 alleles as regards their age, sex, clinical characteristics or laboratory data. This is in agreement with the study by Pehlivan et al. [33].
Whereas the mutant homotype polymorphic TNF-a (AA) was significantly higher in ITP patients conferring almost six-fold increased ITP risk (OR 6.5, 95% CI 2.54– 16.65; P-value 0.000), the A allele was significantly higher in ITP patients than the controls conferring almost twofold increased ITP risk (OR 1.94, 95% CI 1.09–3.45) (Table 2). Plasma TNF-a level in the patient and control groups was not analyzed in the present study. It is known that a substitution at the 308 position of TNF-a resulted in increased production of TNF-a [32]. In conclusion, our study suggests the possibility that TNF-a 308 gene polymorphism contributes to the susceptibility of acquisition of childhood ITP in Egyptian children. Furthermore, TNF-a 308A/A homotype is associated with increased ITP risk, thus signifying the role of TNF-a 308 polymorphism in the pathogenesis of ITP in childhood ITP. Nevertheless, Pehlivan et al. [33] reported that TNF-a 308G/A heterotype predisposes to effective response to medications, and stated that TNF-a 308G/A might be important in the effectiveness of ITP therapy. So, ITP patients with the A allele of TNF-a 308 will benefit the most from blockage of such high-affinity receptor by intravenous infusion of anti-D or they should be selected for the expensive intravenous immunoglobulins therapy [5]. This information is of interest for future study in designing novel therapeutic interventions.
Acknowledgements Conflicts of interest
The authors declare no conflicts of interest.
References 1
2
3
Analysis of the allelic frequencies of the G and A alleles of TNF-a 308 revealed that the G allele in the acute ITP, chronic ITP, and control groups did not differ significantly (OR 0.849, 95% CI 0.640–1.125, P 0.175). Moreover, associations between this polymorphism, and the development and clinical progress of ITP were not observed; this was in accordance with the study by Okulu et al. In contrast, Pehlivan et al. [33] found that the high expression of TNF-a 308G/A phenotype significantly increased in cases with ITP (OR 0.318, 95% CI 0.103– 0.987, P < 0.05).
4
5 6
7 8
9
Blanchette V, Bolton-Maggs P. Childhood immune thrombocytopenic purpura. Diagnosis and management. Hematol Oncol Clin N Am 2010; 24:249–273. Yaprak I, Atabay B, Durak I, Turker M, Oniz H, Ozer EA. Variant clinical courses in children with immune thrombocytopenic purpura: sixteen year experience of a single medical center. Turk J Hematol 2010; 27:147– 155. Bergmann AK, Grace RF, Neufeld EJ. Genetic studies in pediatric ITP: outlook, feasibility, and requirements. Ann Hematol 2010; 89: S95–S103. Anthony RM, Nimmerjahn F, Ashline DJ, Reinhold VN, Paulson JC, Ravetch JV. Recapitulation of IVIG anti-inflammatory activity with a recombinant IgG Fc. Science 2008; 320:373–376. Psaila B, Bussel JB. Immune thrombocytopenic purpura. Hematol Oncol Clin N Am 2007; 21:743–759. Amir A, Gilad O, Yacobovich J, Scheuerman O, Tamary H, Garty BZ. Postvaricella thrombocytopenic purpura. Acta Paediatr 2010; 99:1385– 1388. Akalin E, Murphy B. Gene polymorphisms and transplantation. Curr Opin Immunol 2001; 13:572–576. Terrell DR, Beebe LA, Vesely SK, Neas BR, Segal JB, George JN. The incidence of immune thrombocytopenic purpura in children and adults: a critical review of published reports. Am J Hematol 2010; 85:174–180. Andersson J. Cytokines in idiopathic thrombocytopenic purpura (ITP). Acta Paediatr Suppl 1998; 424:61–64.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Tumor necrosis factor-alpha 308G/A in Egyptian children with ITP El Sissy et al. 463
10
11
12 13
14
15
16
17
18
19 20 21 22
Bidwell JL, Wood NA, Morse HR, Olomolaiye OO, Keen LJ, Laundy GJ. Human cytokine gene nucleotide sequence alignments. Eur J Immunogenet 1999; 26 (Suppl 1):135–223. Foster CB, Zhu S, Erichsen HC, Lehrnbecher T, Hart ES, Choi E, et al. Polymorphisms in inflammatory cytokines and Fcgamma receptors in childhood chronic immune thrombocytopenic purpura: a pilot study. Br J Haematol 2001; 113:596–599. Hajeer AH, Hutchinson IV. Influence of TNF-alpha gene polymorphisms on TNF alpha production and disease. Hum Immunol 2001; 62:1191–1199. Batikhan H, Gokcan MK, Beder E, Akar N, Gerceker OM. Association of the tumor necrosis factor alpha-308G/A polymorphism with nasal polyposis. Eur Arch Otorhinolaryngol 2010; 267:903–908. Elahi MM, Asotra K, Matata BM, Mastana SS. Tumor necrosis factor alpha308 gene locus promoter polymorphism: an analysis of association with health and disease. Biochim Biophys Acta 2009; 1792:163–172. Gonza´lez-Mancho´n C, Ferna´ndez-Pinel M, Arias-Salgado EG, Ferrer M, Alvarez MV, Garcia- Munoz S, et al. Molecular genetic analysis of a compound heterozygote for the glycoprotein (GP) IIb gene associated with Glanzmann’s thrombasthenia: disruption of the 674-687 disulfide bridge in GPIIb prevents surface exposure of GPIIb-IIIa complexes. Blood 1999; 93:866–875. Neunert C, Lim W, Crowther M, Cohen A, Solberg L Jr, Crowther MA. The American Society of Hematology 2011 evidence-based practice guideline for immune thrombocytopenia. Blood 2011; 117:4190–4207. Rodeghiero F, Stasi R, Gernsheimer T, Michel M, Proven D, Arnold DM, et al. Standardization of terminology, definitions and outcome criteria in immune thrombocytopenic purpura of adults and children: report from an International Working Group. Blood 2009; 113:2386–2393. Jrad BB, Chatti A, Laatiri A, Ahmed SB, Romdhane A, Ajimi S, et al. Tumor necrosis factor promoter gene polymorphism associated with increased susceptibility to non-Hodgkin’s lymphomas. Eur J Haematol 2007; 78:117–122. Richardson B. Primer: epigenetics of autoimmunity. Nat Clin Pract Rheumatol 2007; 3:521–527. Cines DB, Blanchette VS. Immune thrombocytopenic purpura. N Engl J Med 2002; 346:995–1008. Beutler B, Grau GE. Tumor necrosis factor in the pathogenesis of infectious diseases. Crit Care Med 1993; 21 (Suppl):S423–S435. Price P, Witt C, Allcock R, Sayer D, Garlepp M, Kok CC, et al. The genetic basis for the association of the 8.1 ancestral haplotype (A1, B8, DR3) with multiple immunopathological diseases. Immunol Rev 1999; 167:257– 274.
23
24
25
26
27
28
29
30
31
32
33
Louis E, Franchimont D, Piron A, Gevaert Y, Schaaf-Lafontaine N, Roland S, et al. Tumour necrosis factor (TNF) gene polymorphism influences TNFalpha production in lipopolysaccharide (LPS)-stimulated whole blood cell culture in healthy humans. Clin Exp Immunol 1998; 113:401–406. Kroeger KM, Carville KS, Abraham LJ. The -308 tumor necrosis factor-alpha promoter polymorphism effects transcription. Mol Immunol 1997; 34:391–399. Teuffel O, Ethier MC, Beyene J, Sung L. Association between tumor necrosis factor-alpha promoter-308A/G polymorphism and susceptibility to sepsis and sepsis mortality: a systematic review and meta-analysis. Crit Care Med 2009; 28:25–29. Settin A, Hassan H, El-Baz R, Hassan T. Association of cytokine gene polymorphisms with psoriasis in cases from the Nile Delta of Egypt. Acta Dermatovenerol Alp Panonica Adriat 2009; 18:105–112. Gambhir D, Lawrence A, Aggarwal A, Misra R, Mandal SK, Naik S. Association of tumor necrosis factor alpha and IL-10 promoter polymorphisms with rheumatoid arthritis in North Indian population. Rheumatol Int 2010; 30:1211–1217. Lin YJ, Chen RH, Wan L, Sheu JC, Huang CM, Lin CW, et al. Association of TNF-{alpha} gene polymorphisms with systemic lupus erythematosus in Taiwanese patients. Lupus 2009; 18:974–979. Vural P, Deg˘irmenciog˘ lu S, Parjldar-Karpuzog˘ lu H, Dog˘ru- Abbasog˘ lu S, Hanagasi HA, Karadag˘ B, et al. The combinations of TNF alpha-308 and IL-6-174 or IL-10-1082 genes polymorphisms suggest an association with susceptibility to sporadic late-onset Alzheimer’s disease. Acta Neurol Scand 2009; 29:10–19. Cerri AP, Arosio B, Viazzoli C, Confalonieri R, Vergani C, Annoni G. The 308(G/A) single nucleotide polymorphism in the TNF-alpha gene and the risk of major depression in the elderly. Int J Geriatr Psychiatry 2009; 28:17–19. Okulu E, ˙Ileri T, C ¸ ulha VK, Aık FM, Eg˘in Y, Uysal Z, et al. The role of tumor necrosis factor-alpha 308 G/A and transforming growth factorbeta 1-915 G/C polymorphisms in childhood idiopathic thrombocytopenic purpura. Turk J Hematol 2011; 28:170–175. Wilson AG, Symons JA, McDowell TL, McDevitt HO, Duff GW. Effects of a polymorphism in the human tumor necrosis factor a promoter on transcriptional activation. Proc Natl Acad Sci USA 1997; 94:3195–3199. Pehlivan M, Okan V, Sever T, Balci SO, Yilmaz M, Babacan T, Pehlıvan S. Investigation of TNF-alpha, TGF-beta 1, IL-10, IL-6, IFN-gamma, MBL, GPIA, and IL1A gene polymorphisms in patients with idiopathic thrombocytopenic purpura. Platelets 2011; 2:588–595.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Tumor necrosis factor-a –308G/A gene polymorphism in Egyptian children with immune thrombocytopenic purpura Maha H. El Sissya, A.H. El Sissyb and Sherif Elanwaryc Immune thrombocytopenic purpura (ITP) is an autoimmune disease characterized by increased platelet destruction. Although the cause of ITP remains unclear, it is accepted that both environmental and genetic factors play an important role in the development of the disease. Children with ITP have a T-helper 1-type cytokine pattern with elevated levels of tumor necrosis factor-alpha (TNF-a) as in most autoimmune diseases. Researchers have shown that polymorphism in the TNF-a gene at position S308 affects gene transcriptions with increased TNF-a production. The current case–control study aimed at detecting the frequency of TNF-a S308G/A gene polymorphism as genetic markers in Egyptian children with ITP, and to clear out their possible role in choosing the treatment protocols of therapy, using PCR restriction fragment length polymorphism assay. Ninety-two ITP patients and 100 age and sex-matched healthy controls were recruited in the study. The results obtained revealed that the frequency of TNF-a S308A/A homotype in ITP patients was significantly higher than that of the controls, and conferred almost six-fold increased risk of ITP acquisition.
The polymorphic A allele frequency was significantly higher in ITP patients than in the controls, conferring almost two-fold increased ITP risk. In conclusion, our study suggests the possibility that TNF-a S308 gene polymorphism may contribute to the susceptibility of childhood ITP in Egyptian children. Blood Coagul Fibrinolysis 25:458–463 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins.
Introduction
(1) Newly diagnosed ITP: cases within 3 months of diagnosis. (2) Persistent ITP: they are the cases between 3 and 12 months of diagnosis and they are the patients who fail to maintain a complete response with treatment or who fail to reach spontaneous remission. (3) Chronic ITP: it continues for more than 12 months. (4) Refractory ITP: the following three criteria are required: (a) loss of remission or failure of remission after splenectomy (b) requirement of treatment to minimize the risk of clinically significant hemorrhage (c) all the other causes of primary ITP and thrombocytopenia have to be eliminated.
Immune thrombocytopenic purpura (ITP) is one of the most frequent causes of thrombocytopenia and is characterized by destruction of autoantibody-mediated platelets. The pathophysiology of ITP is complex and includes antibodies, cytokines, antigen-presenting cells, co-stimulatory molecules, T and B lymphocytes. When autoreactive B lymphocytes produce antiplatelet antibodies, T lymphocytes play an important role in this autoimmune process [1,2]. ITP is an acquired autoimmune disorder and the most common cause of isolated thrombocytopenia in children [3]. Immune thrombocytopenic purpura is caused by the production of antiplatelet antibodies. These autoantibodies opsonize platelets for splenic clearance; resulting in low levels of circulating platelets [4]. These antibodies may also impair platelet production, creating a dual cause of thrombocytopenia [5]. Complete recovery is usually expected within a few days or weeks, regardless of treatment. Nevertheless, therapy is given to patients with ITP because of occasional major bleeding episodes. In approximately 30% of cases, the disease will follow a chronic course with prolonged and recurrent thrombocytopenia [6]. Akalin and Murphy [7] in 2001 classified ITP into the following phases: 0957-5235 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
Blood Coagulation and Fibrinolysis 2014, 25:458–463 Keywords: tumor necrosis factor-alpha, immune thrombocytopenic purpura, genetic polymorphism a
Clinical Pathology Department, bClinical Pathology Department, National Cancer Institute and cPediatric Department, Cairo University, Cairo, Egypt Correspondence to Maha H. El Sissy, MD, Kasr Al aini Hospitals, Cairo University, Cairo, Egypt Tel: +002 01229193373; e-mail: [email protected] Received 9 October 2013 Revised 25 December 2013 Accepted 26 December 2013
It was previously reported that there was an association between the cytokine gene polymorphisms affecting the cytokine production and secretion and infectious diseases, allergic diseases, autoimmune diseases, and malignant diseases, both at the stage of formation of disease and in the course of disease and their responses to treatment [7]. Population-based studies have shown that ITP has an incidence of up to 6.4 per 100 000 children and 3.3 per 100 000 adults per year [8]. DOI:10.1097/MBC.0000000000000089
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Tumor necrosis factor-alpha 308G/A in Egyptian children with ITP El Sissy et al. 459
Children with ITP have a T-helper 1 (Th1)-type cytokine pattern with elevated levels of tumor necrosis factoralpha (TNF-a), as in most autoimmune diseases [9]. Researchers have shown that polymorphism in the TNF-a gene at position 308 affect gene transcription with increased TNF-a production [10]. The transcriptionally more active allele of TNF-a (allele 2 of 308) was less frequently observed in pediatric chronic ITP patients than in healthy controls [11]. Tumor necrosis factor-alpha is a pleiotropic cytokine produced primarily by macrophages and T cells, and has a range of inflammatory and immunomodulatory activity [12]. Polymorphisms of TNF-a promoter are associated with high levels of TNF-a and have been studied as a determinant of susceptibility to numerous diseases [13]. A study that examined inflammatory cytokines and Fcg receptor (FcgR) polymorphisms in chronic childhood ITP reported that polymorphisms in two low-affinity FcgR genes (IIIA and IIIB) and two pro-inflammatory cytokine genes [TNF-a and lymphotoxin-alpha (LTA)] were associated with chronic childhood ITP [11]. A single-base polymorphism within the promoter region of TNF gene at nucleotide position 308 results in two allelic forms: one with guanine, TNF-G allele (TNF1 allele), and the other, in which guanine is substituted by adenosine, TNF-A allele (TNF2 allele). TNF-A allele has been found to correlate with enhanced spontaneous or stimulated TNF production in vivo and in vitro [14]. The current case–control study aimed at detecting the frequency of TNF-a 308G/A gene polymorphism in Egyptian children with ITP as genetic markers for ITP risk, and to clear out their possible role in choosing the treatment protocols of ITP patients.
Patients and methods The present study was conducted on 92 ITP patients (12 acute and 80 chronic) attending the Haematology Clinic of the Children Hospital, Faculty of Medicine, Cairo University, as well as 100 age and sex-matched healthy children as a control group; all patients and control groups gave a written consent accepting research aid and our case–control study was approved by the ethical committee of the Clinical Pathology Department, Cairo University. Diagnosis of ITP was based on proper history taking with special attention to history of intake of medications known to induce signs of ITP as petechiae, ecchymosis or mucous membrane bleeding, and to exclude signs of concomitant autoimmune disorders such as juvenile rheumatoid arthritis, systemic lupus erythematosus (SLE), and vitiligo. Laboratory assessment included complete blood count revealing isolated thrombocytopenia) platelet count 12 months’ duration) [17]. The study design was approved by the Scientific Research Committee of Clinical Pathology and Pediatrics Departments, Faculty of Medicine, Cairo University. All parents and/or surrogates were informed about the aims of the study. Formal written consents were obtained from the caregivers of the participants who agreed to their child’s participation in the study.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
460 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 5
Detection of tumor necrosis factor-alpha S308 genetic polymorphism by PCR restriction fragment length polymorphism assay
Five milliliters venous blood was withdrawn under complete aseptic conditions from all participants on EDTA. Samples were either stored in the same vacutainer at 208C or used directly within 24 h for DNA extraction. DNA was extracted from whole blood using AxyPrep Blood Genomic DNA Miniprep Kit (Axygen Biosciences, Union City California, USA). Genotyping was based upon the methods described by Jrad et al. [18] as the target genes were amplified by PCR then restriction with the endonuclease NcoI. All PCR reactions were performed in a total volume of 25 ml containing 12.5 ml Master mix (Bioron GmbH, Ludwigshafen Germany), 1 ml forward primer (25 pmol), 1 ml reverse primer (25 pmol), 5.5 ml nuclease-free water and 5 ml genomic extracted DNA. For TNF-a 308 genotyping, the following primers were used: forward primer 50 -AGGCAATAGGTTTTGAGG GCCAT-30 , and reverse primer 50 -TCCTCCCTGCT CCGATTCCG-30 (Fermentas, Lithuania). The thermocycler program applied was heating at 958C for 5 min, followed by 29 cycles of denaturation at 958C for 30 s, annealing at 608C for 30 s, and extension at 728C for 45 s. A final extension step was carried out at 728C for 10 min. The PCR product (107 bp) was digested with NcoI (Fermentas, Cat. No. #ER0571). The product was visualized by 3% agarose gel electrophoresis stained with ethidium bromide under ultraviolet (UV) light. A DNA molecular weight marker was also run to identify the site of bands. The presence of NcoI restriction site in the mutant allele of TNF-a (A allele) was indicated by the cleavage of the 107 bp amplicon to yield two fragments of 87 and 20 bp (Fig. 1).
data were expressed as mean and SD or median and range as appropriate. Qualitative data were expressed as frequency and percentage. Chi-squared test (Fisher’s exact test) was used to examine the relation between qualitative variables. For quantitative data, comparison between two groups was done using either Student’s t-test or Mann–Whitney test (nonparametric t-test). Comparison between three groups was done using Kruskal–Wallis test [nonparametric analysis of variance (ANOVA)]. Odds ratio (OR) and 95% confidence intervals (CIs) were used for risk estimation. Kappa test was used to evaluate agreement between two genotypes. A P-value less than 0.05 was considered significant.
Results The study included 92 ITP cases. They were 48 females (52.2%) and 44 males (47.8%). Their ages ranged between 1 and 14 years with a mean value of age 8.26 4.49 years. Twelve patients (12/92 13.1%) were acute and 80 of 92 (86.9%) were chronic cases; clinical and laboratory data of ITP patients are summarized in Table 1. One hundred age and sex-matched unrelated healthy individuals were included in the current study as a control group. They were 45 females (45%) and 55 males (55%). Their ages ranged between 1 and 13 years with mean value of 6.18 3.494 years. Indirect antiplatelet antibodies were detected in three patients with acute ITP (3/12–25%) and 20 patients (20/80–25%) with chronic ITP, revealing no statistically significant difference between acute and chronic cases.
Data were analyzed using IBM SPSS advanced statistical version 20 (SPSS Inc., Chicago, Illinois, USA). Numerical
The frequency of TNF-a 308A/A homotype in ITP patients was significantly higher than that of the controls, and conferred almost six-fold increased risk of ITP acquisition (OR 6.5, 95% CI 2.54–16.65). The frequency of the combined polymorphic genotypes – TNF-a 308G/A heterotype and A/A homotype – was
Fig. 1
Table 1
Statistical analysis
Descriptive data of ITP patients
Item Sex Bleeding Spleen
Degree of anemia
Degree of thrombocytopenia
Identification of the TNF-308 genotypes by agarose gel electrophoresis. M: 50–1000 Ladder size marker. The 20-bp band is not visible in this photograph. Lanes 3 and 5: TNF-308 GG genotype (wild type) showing a 107-bp band. Lane 1: TNF-308 AA homotype showing 87-bp band. Lanes 2 and 4: TNF-308 GA heterotype showing 87 and 107-bp bands. TNF, tumor necrosis factor.
History of viral infections or immunization Family history Type of ITP Therapy
Male Female Positive Enlarged Normal Splenectomy Mild Moderate Marked Hb: 11gm% Moderate Marked Positive Negative Negative Acute Chronic Steroids
Frequency
Percentage
44/92 48/92 92/92 4/92 79/92 9/92 80/92 8/92 3/92 1/92 6/92 86/92 31/92 61/92 92/92 12/92 80/92 92/92
47.8 52.2 100 4.3 85.9 9.8 87 8.7 3.3 1.1 6.5 93.5 33.7 66.3 100 13 87 100
Hb, haemoglobin; ITP, immune thrombocytopenic purpura.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Tumor necrosis factor-alpha 308G/A in Egyptian children with ITP El Sissy et al. 461
Table 2
Genotypes and alleles frequency of TNF-a S308 polymorphism in ITP cases and controls
TNF-a 308 Wild genotype (G/G) Heterotype (G/A) Homotype (A/A) Combined polymprphic genotypes (G/A–A/A) G wild-type allele A polymorphic allele
Cases (n ¼ 92)
Control (n ¼ 100)
43/92 22/92 27/92 49/92 86 (0.54) 98 (0.38)
63/100 31/100 6/100 37/100 128 (0.80) 72 (0.195)
OR (95% CI) 1.439 0.849 6.5 1.94 1.94
(1.046–1.981) (0.640–1.125) (2.54–16.65) (1.09–3.45) (1.09–3.45)
P-value 0.017 0.175 0.001 0.024 0.001
CI, confidence interval; ITP, immune thrombocytopenic purpura; OR, odds ratio; TNF-a, tumor necrosis factor-alpha.
significantly higher in ITP cases than in controls (OR 1.94, 95% CI 1.09–3.45, P-value 0.024).
platelet antigen (HPA) systems, were associated with the development of ITP and response to treatment [20].
Also the polymorphic A allele frequency was significantly higher in ITP patients than the controls conferring almost two-fold increased ITP risk (OR 1.94, 95% CI 1.09–3.45) (Table 2), suggesting its possible role as a genetic risk factor for tendency of childhood ITP.
Tumor necrosis factor-alpha is a critical cytokine in the inflammatory response to infection. Accordingly, any genetic variability in the production of TNF-a after an infectious stimulus could have a significant impact on the degree of inflammatory response and therefore potentially influence the clinical outcome [21].
Statistical comparison between ITP patients with the wild genotype and those with polymorphic genotypes of TNFa 308 gene revealed that the frequency of the polymorphic genotype was significantly lower in males and lactate dehydrogenase level was significantly lower among ITP patients harboring the polymorphic A allele. Otherwise, there was no statistically significant difference noticed between the two patient groups, as regards the degree of thrombocytopenia, bleeding, splenomegaly, or their response to treatment (data not shown). The frequency of TNF-a 308G/A heterotype and A/A homotype in acute ITP patients was not significantly different from chronic ITP (OR 1.481, 95% CI 0.604– 3.635 and OR 0.833, 95% CI 0.296–2.347, respectively); also, the polymorphic A allele frequency was not significantly different in acute than chronic ITP patients (Table 3).
Discussion Although the cause of ITP remains unclear, it is generally accepted that both environmental and genetic factors play an important role in the development of the disease [19]. Many studies have focused on the association between some cytokine gene polymorphisms and susceptibility to ITP. Studies that investigated genetic risk factors in the pathogenesis of ITP reported that polymorphisms of human leucocyte antigen class II antigens, TNF-a, transforming growth factor-b, and human Table 3
Significant evidence supports the biologic importance of polymorphisms within the TNF-a promoter region. A guanine (G) to adenine (A) transition at TNF-a 308 is perhaps the best studied cytokine polymorphism and the one for which the best evidence of functional significance exists [22]. Stimulation studies in healthy volunteers suggested that carriage of the TNF-a 308 A allele is associated with significantly greater TNF-a production [23] and TNF-a mRNA transcription [24]. Tumor necrosis factor-alpha is a potent immunomediator and pro-inflammatory cytokine shown in the pathogenesis of many human diseases. In their meta-analysis, Teuffel et al. [25] indicated that TNF-a 308A/G polymorphism was associated with sepsis. Settin et al. [26] found that the frequency of TNF-a 308G/A genotype increased in patients with psoriasis and they determined the G/G frequency to be low. Gambhir et al. [27] reported that the frequency of TNF-a 308 A genotype was lower in 222 cases with rheumatoid arthritis in the northern section of India than in 208 healthy control cases, and that the presence of this genotype was protective against the disease. In their study in Taiwan, Lin et al. [28] detected the frequency of allele A in TNF-a 308 polymorphisms as significantly high in cases with malar rash, discoid rash, photosensitivity, oral ulcer, and serositis compared to the control cases in SLE patients, and they demonstrated that in the Taiwanese patient population,
Risk genotypes and alleles frequency of TNF-a S308 polymorphism in acute and chronic ITP patients
TNF-a 308 Wild genotype (G/G) Heterotype (G/A) Homotype (A/A) G wild-type allele A polymorphic allele
Acute ITP (n ¼ 12)
Chronic ITP (n ¼ 80)
5/12 4/12 3/12 10 (0.58) 14 (0.41)
38/80 18/80 24/80 76 (0.58) 84 (0.41)
OR (95% CI) 1.111 1.481 0.833 1.010
(0.659–1.872) (0.604–3.635) (0.296–2.347) (0.608–1.689)
P-value 0.475 0.311 0.509 0.569
CI, confidence interval; ITP, immune thrombocytopenic purpura; OR, odds ratio; TNF-a, tumor necrosis factor-alpha.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
462 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 5
TNF-a 308 polymorphism played a role in susceptibility to the formation of SLE. Vural et al. [29] showed that the frequency of TNF-a 308 gene polymorphisms increased 6.5-fold in sporadic late-onset Alzheimer’s disease compared to the controls. Cerri et al. [30] demonstrated that in elderly patients with major depression, TNF-a 308G/A genotype contributed to the development of major depression. To the best of our knowledge, the role of TNF-a 308A/ A homotype polymorphism in the development of ITP has not been previously studied in Africans. In the present study, the genotype frequency of TNF-a 308 mutant genotypes G/A heterotype and A/A homotype polymorphism was evaluated in terms of their correlation with susceptibility to the development of ITP. In the current study, genotyping of TNF-a 308 gene by PCR restriction fragment length polymorphism (PCR-RFLP) assay revealed that the wild-type alleles were detected in 46.8%, whereas the mutant genotypes of TNF-a 308G/A heterotype and A/A homotype were detected in 23.9 and 29.3% of the patients, respectively. The frequency of the mutant genotypes in Egyptian ITP patients were different to that reported in the study by Okulu et al. [31] (36%) in Turkish populations. The frequency of TNF-a 308G/A heterotype in ITP patients was near to that of the control individuals (22 versus 31%). This was not in accordance with the study by Okulu et al. who reported (36 versus 22.9%) ITP patients versus control individuals, respectively. The frequency of TNF-a 308A/A homotype in ITP patients was not close to that of the control individuals (27 versus 6%). This was not in accordance with the study by Okulu et al. who reported (18 versus 22.9%) ITP patients versus control individuals, respectively. No statistically significant difference was noticed between ITP patients harboring the normal or polymorphic TNF-a 308 alleles as regards their age, sex, clinical characteristics or laboratory data. This is in agreement with the study by Pehlivan et al. [33].
Whereas the mutant homotype polymorphic TNF-a (AA) was significantly higher in ITP patients conferring almost six-fold increased ITP risk (OR 6.5, 95% CI 2.54– 16.65; P-value 0.000), the A allele was significantly higher in ITP patients than the controls conferring almost twofold increased ITP risk (OR 1.94, 95% CI 1.09–3.45) (Table 2). Plasma TNF-a level in the patient and control groups was not analyzed in the present study. It is known that a substitution at the 308 position of TNF-a resulted in increased production of TNF-a [32]. In conclusion, our study suggests the possibility that TNF-a 308 gene polymorphism contributes to the susceptibility of acquisition of childhood ITP in Egyptian children. Furthermore, TNF-a 308A/A homotype is associated with increased ITP risk, thus signifying the role of TNF-a 308 polymorphism in the pathogenesis of ITP in childhood ITP. Nevertheless, Pehlivan et al. [33] reported that TNF-a 308G/A heterotype predisposes to effective response to medications, and stated that TNF-a 308G/A might be important in the effectiveness of ITP therapy. So, ITP patients with the A allele of TNF-a 308 will benefit the most from blockage of such high-affinity receptor by intravenous infusion of anti-D or they should be selected for the expensive intravenous immunoglobulins therapy [5]. This information is of interest for future study in designing novel therapeutic interventions.
Acknowledgements Conflicts of interest
The authors declare no conflicts of interest.
References 1
2
3
Analysis of the allelic frequencies of the G and A alleles of TNF-a 308 revealed that the G allele in the acute ITP, chronic ITP, and control groups did not differ significantly (OR 0.849, 95% CI 0.640–1.125, P 0.175). Moreover, associations between this polymorphism, and the development and clinical progress of ITP were not observed; this was in accordance with the study by Okulu et al. In contrast, Pehlivan et al. [33] found that the high expression of TNF-a 308G/A phenotype significantly increased in cases with ITP (OR 0.318, 95% CI 0.103– 0.987, P < 0.05).
4
5 6
7 8
9
Blanchette V, Bolton-Maggs P. Childhood immune thrombocytopenic purpura. Diagnosis and management. Hematol Oncol Clin N Am 2010; 24:249–273. Yaprak I, Atabay B, Durak I, Turker M, Oniz H, Ozer EA. Variant clinical courses in children with immune thrombocytopenic purpura: sixteen year experience of a single medical center. Turk J Hematol 2010; 27:147– 155. Bergmann AK, Grace RF, Neufeld EJ. Genetic studies in pediatric ITP: outlook, feasibility, and requirements. Ann Hematol 2010; 89: S95–S103. Anthony RM, Nimmerjahn F, Ashline DJ, Reinhold VN, Paulson JC, Ravetch JV. Recapitulation of IVIG anti-inflammatory activity with a recombinant IgG Fc. Science 2008; 320:373–376. Psaila B, Bussel JB. Immune thrombocytopenic purpura. Hematol Oncol Clin N Am 2007; 21:743–759. Amir A, Gilad O, Yacobovich J, Scheuerman O, Tamary H, Garty BZ. Postvaricella thrombocytopenic purpura. Acta Paediatr 2010; 99:1385– 1388. Akalin E, Murphy B. Gene polymorphisms and transplantation. Curr Opin Immunol 2001; 13:572–576. Terrell DR, Beebe LA, Vesely SK, Neas BR, Segal JB, George JN. The incidence of immune thrombocytopenic purpura in children and adults: a critical review of published reports. Am J Hematol 2010; 85:174–180. Andersson J. Cytokines in idiopathic thrombocytopenic purpura (ITP). Acta Paediatr Suppl 1998; 424:61–64.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Tumor necrosis factor-alpha 308G/A in Egyptian children with ITP El Sissy et al. 463
10
11
12 13
14
15
16
17
18
19 20 21 22
Bidwell JL, Wood NA, Morse HR, Olomolaiye OO, Keen LJ, Laundy GJ. Human cytokine gene nucleotide sequence alignments. Eur J Immunogenet 1999; 26 (Suppl 1):135–223. Foster CB, Zhu S, Erichsen HC, Lehrnbecher T, Hart ES, Choi E, et al. Polymorphisms in inflammatory cytokines and Fcgamma receptors in childhood chronic immune thrombocytopenic purpura: a pilot study. Br J Haematol 2001; 113:596–599. Hajeer AH, Hutchinson IV. Influence of TNF-alpha gene polymorphisms on TNF alpha production and disease. Hum Immunol 2001; 62:1191–1199. Batikhan H, Gokcan MK, Beder E, Akar N, Gerceker OM. Association of the tumor necrosis factor alpha-308G/A polymorphism with nasal polyposis. Eur Arch Otorhinolaryngol 2010; 267:903–908. Elahi MM, Asotra K, Matata BM, Mastana SS. Tumor necrosis factor alpha308 gene locus promoter polymorphism: an analysis of association with health and disease. Biochim Biophys Acta 2009; 1792:163–172. Gonza´lez-Mancho´n C, Ferna´ndez-Pinel M, Arias-Salgado EG, Ferrer M, Alvarez MV, Garcia- Munoz S, et al. Molecular genetic analysis of a compound heterozygote for the glycoprotein (GP) IIb gene associated with Glanzmann’s thrombasthenia: disruption of the 674-687 disulfide bridge in GPIIb prevents surface exposure of GPIIb-IIIa complexes. Blood 1999; 93:866–875. Neunert C, Lim W, Crowther M, Cohen A, Solberg L Jr, Crowther MA. The American Society of Hematology 2011 evidence-based practice guideline for immune thrombocytopenia. Blood 2011; 117:4190–4207. Rodeghiero F, Stasi R, Gernsheimer T, Michel M, Proven D, Arnold DM, et al. Standardization of terminology, definitions and outcome criteria in immune thrombocytopenic purpura of adults and children: report from an International Working Group. Blood 2009; 113:2386–2393. Jrad BB, Chatti A, Laatiri A, Ahmed SB, Romdhane A, Ajimi S, et al. Tumor necrosis factor promoter gene polymorphism associated with increased susceptibility to non-Hodgkin’s lymphomas. Eur J Haematol 2007; 78:117–122. Richardson B. Primer: epigenetics of autoimmunity. Nat Clin Pract Rheumatol 2007; 3:521–527. Cines DB, Blanchette VS. Immune thrombocytopenic purpura. N Engl J Med 2002; 346:995–1008. Beutler B, Grau GE. Tumor necrosis factor in the pathogenesis of infectious diseases. Crit Care Med 1993; 21 (Suppl):S423–S435. Price P, Witt C, Allcock R, Sayer D, Garlepp M, Kok CC, et al. The genetic basis for the association of the 8.1 ancestral haplotype (A1, B8, DR3) with multiple immunopathological diseases. Immunol Rev 1999; 167:257– 274.
23
24
25
26
27
28
29
30
31
32
33
Louis E, Franchimont D, Piron A, Gevaert Y, Schaaf-Lafontaine N, Roland S, et al. Tumour necrosis factor (TNF) gene polymorphism influences TNFalpha production in lipopolysaccharide (LPS)-stimulated whole blood cell culture in healthy humans. Clin Exp Immunol 1998; 113:401–406. Kroeger KM, Carville KS, Abraham LJ. The -308 tumor necrosis factor-alpha promoter polymorphism effects transcription. Mol Immunol 1997; 34:391–399. Teuffel O, Ethier MC, Beyene J, Sung L. Association between tumor necrosis factor-alpha promoter-308A/G polymorphism and susceptibility to sepsis and sepsis mortality: a systematic review and meta-analysis. Crit Care Med 2009; 28:25–29. Settin A, Hassan H, El-Baz R, Hassan T. Association of cytokine gene polymorphisms with psoriasis in cases from the Nile Delta of Egypt. Acta Dermatovenerol Alp Panonica Adriat 2009; 18:105–112. Gambhir D, Lawrence A, Aggarwal A, Misra R, Mandal SK, Naik S. Association of tumor necrosis factor alpha and IL-10 promoter polymorphisms with rheumatoid arthritis in North Indian population. Rheumatol Int 2010; 30:1211–1217. Lin YJ, Chen RH, Wan L, Sheu JC, Huang CM, Lin CW, et al. Association of TNF-{alpha} gene polymorphisms with systemic lupus erythematosus in Taiwanese patients. Lupus 2009; 18:974–979. Vural P, Deg˘irmenciog˘ lu S, Parjldar-Karpuzog˘ lu H, Dog˘ru- Abbasog˘ lu S, Hanagasi HA, Karadag˘ B, et al. The combinations of TNF alpha-308 and IL-6-174 or IL-10-1082 genes polymorphisms suggest an association with susceptibility to sporadic late-onset Alzheimer’s disease. Acta Neurol Scand 2009; 29:10–19. Cerri AP, Arosio B, Viazzoli C, Confalonieri R, Vergani C, Annoni G. The 308(G/A) single nucleotide polymorphism in the TNF-alpha gene and the risk of major depression in the elderly. Int J Geriatr Psychiatry 2009; 28:17–19. Okulu E, ˙Ileri T, C ¸ ulha VK, Aık FM, Eg˘in Y, Uysal Z, et al. The role of tumor necrosis factor-alpha 308 G/A and transforming growth factorbeta 1-915 G/C polymorphisms in childhood idiopathic thrombocytopenic purpura. Turk J Hematol 2011; 28:170–175. Wilson AG, Symons JA, McDowell TL, McDevitt HO, Duff GW. Effects of a polymorphism in the human tumor necrosis factor a promoter on transcriptional activation. Proc Natl Acad Sci USA 1997; 94:3195–3199. Pehlivan M, Okan V, Sever T, Balci SO, Yilmaz M, Babacan T, Pehlıvan S. Investigation of TNF-alpha, TGF-beta 1, IL-10, IL-6, IFN-gamma, MBL, GPIA, and IL1A gene polymorphisms in patients with idiopathic thrombocytopenic purpura. Platelets 2011; 2:588–595.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
